一种来自乳酸乳球菌的新型氨肽酶A的制备及特性分析

Production and characterization of a novel aminopeptidase A from Lactococcus lactis

摘要:

氨肽酶A (aminopeptidase A,Pep A)能特异性地水解N末端为谷氨酸(glutamic acid,Glu)或天冬氨酸(aspartic acid,Asp)的肽链，提高蛋白质的水溶性和食物的风味，在食品工业和肉类加工中具有一定的应用前景。本研究采用全基因合成的方式获得了乳酸乳球菌(Lactococcus lactis ssp.lactis) IL1403氨肽酶A (Lactococcus lactis-Pep A,Lc-Pep A)的编码基因，将该基因克隆并导入毕赤酵母(Pichia pastoris) GS115(His4)，在毕赤酵母中实现了Lc-Pep A的高效分泌表达，表达产物经鉴定和纯化制备后，进行了生物学特性的分析。结果表明，Lc-Pep A具有较强的底物特异性，对2种底物谷氨酸对硝基苯胺(glutamic acid-p-nitroaniline,Glu-pNA)和天冬氨酸对硝基苯胺(aspartic acid-p-nitroaniline,Asp-pNA)具有相似的催化活力和酶动力学参数。Lc-Pep A是一种金属蛋白酶，最适反应温度为60℃，最适pH为8.0，具有较宽的热稳定性和酸碱稳定性。金属离子Co²⁺、Mn²⁺及Zn²⁺等对酶活力具有不同程度的激活作用，而Ni²⁺和Cu²⁺对酶活力具有强烈的抑制作用。Lc-Pep A对常规蛋白酶抑制剂不敏感，但能被金属蛋白酶抑制剂、EDTA及二硫键还原剂抑制。这些研究为Lc-Pep A的生产和指导该酶的应用打下了坚实的基础。

Abstract:

Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co²⁺, Mn²⁺, and Zn²⁺ but was strongly inhibited by Ni²⁺and Cu²⁺. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.